Microfluidic device for generating neural cells to simulate post-stroke conditions

ABSTRACT

This application provides devices for modeling ischemic stroke conditions. The devices can be used to culture neurons and to subject a first population of the neurons to low-oxygen conditions and a second population of neurons to normoxic conditions. The neurons are cultured on a porous barrier, and on the other side of the barrier run one or more fluid-filled channels. By flowing fluid with different oxygen levels through the channels, one can deliver desired oxygen concentrations to the cells nearest those channels.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 13/758,572, filed on Feb. 4, 2013 and titled“MICROFLUIDIC DEVICE FOR GENERATING NEURAL CELLS TO SIMULATE POST-STROKECONDITIONS,” which claims priority to U.S. Provisional PatentApplication No. 61/594,668, filed Feb. 3, 2012. Each of the foregoingapplications is incorporated herein by reference in their entirety.

BACKGROUND OF THE DISCLOSURE

Ischemic stroke occurs when neural tissue is damaged due to low oxygenlevels. Magnetic resonance imaging detects three distinct areas in thebrain of a stroke patient. The first, called the core, is the area thatreceives little or no oxygen, and experiences severe necrosis. A secondarea consists of normal tissue that receives sufficient oxygen. Thethird area falls between the core and the normal tissue, and is calledthe penumbra. Functionally, the penumbral area has compromised bloodflow (CBF) and decreased oxygen consumption (CMRO₂), which translatesinto neurons that are still viable but stressed. Over the course ofseveral days following a stroke, the core gradually expands into thepenumbra. Treatments for ischemic stroke are extremely limited, in partbecause of the lack of adequate in vitro models for ischemic stroke.There is a need in the art for an ischemic stroke model that allows onenot only to study the basic biology of the core and penumbra but to testnovel therapeutic interventions.

SUMMARY OF THE DISCLOSURE

This application provides devices for modeling ischemic strokeconditions. The devices can be used to culture neurons and to subject afirst population of the neurons to low-oxygen conditions and a secondpopulation of neurons to normoxic conditions. The neurons are culturedon a porous barrier, and on the other side of the barrier run one ormore fluid-filled channels. By flowing fluid with different oxygenlevels through the channels, one can deliver desired oxygenconcentrations to the cells nearest those channels. The devices hereinprovide in vitro models for a number of in vivo medical conditions.

According to one aspect of the disclosure, a device for modelingischemic stroke conditions includes a fluid-containing chamber; a porousbarrier to which the cells are adhered; and a first channel having aninlet and an outlet, separated from the chamber and cells by the porousbarrier, wherein the cells comprise a first population that is proximalto the first channel and a second population that is distal from thefirst channel. The device also includes a first array of electrodespositioned between the first channel and the porous barrier and alignedalong the length of the first channel; and a second array of electrodescoupled to the fluid facing side of the roof of the fluid-containingchamber.

In certain implementations, the fluid-containing chamber includes cells.The cells can include central nervous system cells, cardiac musclecells, or tumor cells. The central nervous system cells can includeneurons, microglia, astrocytes, oligodendrocytes, or neural progenitors.In other implementations, the cells include a monolayer of muscle cellsor endothelial cells along at least one wall of the channel. In yetother implementations, the cells are organized as a three-dimensionalculture. In some implementations, the first population of cells are lessthan 100 microns apart. In certain implementations, immune cells aredisposed in the channel.

In some implementations, the first channel is adapted for flowing fluidalong the barrier, and the cells experience substantially no shearstress when fluid flows through the first channel. In otherimplementations, the electrodes are between about 100 μm and 150 μm indiameter and configured to measure trans-endothelial electricalresistance.

In yet other implementations, the device also includes a second channelconfigured for fluid flow, wherein the porous barrier separates thesecond channel from the chamber and the cells. In some of theseimplementations, the first channel contains a first fluid and the secondchannel contains a second fluid. The first fluid and the second fluidcan have different levels of oxygen or oxygen scavenger. In someimplementations, the flow of fluid through the first and second channelis independently controllable. The porous barrier is sufficientlytransparent to allow microscopy of the cells in some implementations. Insome implementations, the device further includes a proteinaceouscoating that is adhered to the porous membrane.

According to another aspect of the disclosure, a for modeling ischemicstroke conditions includes a cellular chamber made of a materialsuitable for cell culture; a base comprising a channel, wherein thechannel has an inlet and an outlet, and the channel is sized to beproximal to a first region of the cellular chamber and distal from asecond region of the cellular chamber; a porous membrane suitable as asubstrate for cell culture, wherein the porous membrane is sized toseparate the channel from the cellular chamber; a plurality ofelectrodes configured to measure trans-endothelial electricalresistance; and a means for securing the microporous membrane betweenthe chamber and the channel.

In some implementations, the kit also includes a trans-endothelialelectrical resistance module, an atmospheric control system and/or acontroller.

According to one aspect of the disclosure, a method for modelingischemic conditions includes providing a microfluidic device including afluid-filled chamber, a channel, a porous barrier separating the chamberfrom the channel, a first population of CNS cells proximal to thechannel, and a second population of CNS cells distal from the channel.The first population of the cells is then exposed to a low concentrationof oxygen, and the second population of the cells is exposed to a higherconcentration of oxygen; thereby modeling the ischemic condition. Themethod also includes measuring at least one cellular property, such as acellular factor.

In some implementations, the ischemic condition is ischemic stroke andthe cells can include central nervous system cells such as neurons,microglia, astrocytes, oligodendrocytes, or neural progenitors.

In other implementations, a third population of CNS cells is disposedbetween the first population and the second population, and the thirdpopulation of CNS cells models a penumbra produced by an ischemicstroke.

In certain implementations, the method also includes measuringtrans-endothelial electrical resistance across the cells, visualizingthe cells in the device by microscopy, and/or removing the cells fromthe device and performing biochemical analysis or microscopy on theremoved cells.

In some implementations, the method of measuring the at least onecellular property further includes testing for a factor secreted by thecells. The factor can be measured from the chamber and/or one of thechannels.

In other implementations, exposing the first population of cells to alower concentration of oxygen includes delivering an oxygen scavengerthrough the channel within an effective distance of the first populationof cells. Exposing the second population of cells to a highconcentration of oxygen includes delivering oxygenated fluid through thechannel within an effective distance of the second population of cells.

The cells are exposed to a test agent in some implementations. The testagent can be selected such that it promotes neurodegeneration, necrosis,or apoptosis. In other implementations, the agent is a cancer cellcapable of invading neural tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing will be provided by the Office upon request and payment ofnecessary fee.

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the described implementations may be shownexaggerated or enlarged to facilitate an understanding of the describedimplementations. In the drawings, like reference characters generallyrefer to like features, functionally similar and/or structurally similarelements throughout the various drawings. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the teachings. The drawings are not intended to limitthe scope of the present teachings in any way. The system and method maybe better understood from the following illustrative description withreference to the following drawings in which:

FIG. 1A is a schematic illustration of a system for modeling ischemicstroke; according to one implementation of the disclosure.

FIG. 1B illustrates an exploded view of an ischemic model device suchthat can be used in the system of FIG. 1A; according to oneimplementation of the disclosure.

FIG. 2 is a side view of an ischemic model device comprising CNS cells;according to one implementation of the disclosure.

FIG. 3 illustrates a base of an ischemic model device containingchannels with capillary-like geometry; according to one implementationof the disclosure.

FIG. 4A is a side view of an ischemic model device including CNS cellsin the cell culture chamber and endothelial cells and an immune cell inthe channel; according to one implementation of the disclosure.

FIG. 4B is an isometric view of the base layer of an ischemic modeldevice; according to one implementation of the disclosure.

FIG. 5 is a flow chart of a method for modeling ischemic stroke using asystem similar to the system of FIG. 1; according to one implementationof the disclosure.

FIG. 6 illustrates three ischemic model devices arranged in parallel;according to one implementation of the disclosure.

FIG. 7 illustrates a suitable oxygen gradient for the ischemic modeldevice. The x and y labels refer to the coordinates of the device inmeters.

FIG. 8 is as photograph of an ischemic stroke model device with twochannels.

FIGS. 9A-F illustrates fluorescence microscopy images of neural cellscultured in an ischemic stroke model device.

FIGS. 10A and B depict the calcium levels in neural cells exposed to apotassium gradient. Panel A is a series of images gathered in a timecourse experiment. The line in the t=20 s panel represents 50 μm. PanelB shows the regions of interest (ROI) mapped onto a photograph of thecell culture (upper panel). The scale on right indicates the change influorescence intensity (510 nm, emission; 380 nm, excitation), withhigher intensity representing higher [Ca²⁺]i. The relative [Ca²⁺]ilevels at each region of interest over time are graphed in the lowerpanel with ROI #1 representing the ROI at the upper right and ROI #11representing the ROI at the lower left.

DETAILED DESCRIPTION

The various concepts introduced above and discussed in greater detailbelow may be implemented in any of numerous ways, as the describedconcepts are not limited to any particular manner of implementation.Examples of specific implementations and applications are providedprimarily for illustrative purposes.

Ischemic stroke produces a region of tissue at the site of the ischemicregion, called the core, that includes necrotic neural tissue.Surrounding the core is the penumbra, a region of neural tissue that isless strongly affected by the disrupted oxygenation. One of the goals ofstroke therapy is to prevent expansion of the core by enhancing survivalof cells in the penumbra. To date, only a single drug has been approvedfor the treatment of acute ischemic stroke. This drug, tissueplasminogen activator (tPA), is subject to significant limitations thatresult in less than 3% of stroke victims receiving this thrombolytic. Amajor impediment in the development of novel drug therapies forminimizing stroke injury is that the mechanisms that contribute toprogression of the stroke lesion remain poorly understood. Moreover, theinteraction between cells in the ischemic core, penumbra, and normalregions have not been mimicked or studied sufficiently in real time tofully comprehend how these interactions influence expansion of thelesion.

This disclosure provides a model device that generates accurate ischemicconditions in vitro to facilitate fundamental understanding of cellularcommunication between necrotic core and the ischemic penumbra. Thedevice is also suitable as screening tool for therapeutic interventions.In particular, this application discloses a device that generatesnormoxic, anoxic, and hypoxic environments closely communicating witheach other to replicate the in vivo conditions that occur duringischemic stroke.

I. Non-Cell Components of the Ischemic Stroke Model

FIG. 1A illustrates a system 100 for modeling ischemic stroke. Thesystem 100 includes an atmospheric control system (ACS) 120. Within theACS 120 the system includes at least one camera 130 and at least oneischemic model device 110. The ACS 120 also includes a plurality ofaccess ports 121 and pressure valves 122. A first set of pumps 140 flowfluid from at least one fluid source 150 through the plurality ofischemic model devices 110. A second series of pumps 140 flow gas from agas source 160 into the ACS 120. System 100 also includes atrans-endothelial electrical resistance (TER) module 170. The system 100further includes a controller 180 with at least one processor 181 forcontrolling the components of system 100.

Referring to system 100, and in greater detail, the ACS 120 maintainsproper atmospheric conditions for ischemic stroke experimentation. Insome implementations, the ACS 120 maintains a specific experimentaltemperature, pressure, gas mixture, humidity, or any combination thereofwithin the testing area. The ACS 120 is connected to a plurality of gassources 160. To maintain specific gas mixtures, the ACS 120 pumps gasesinto the ACS 120 with a pump 140. The ACS 120 also controls a pressurevalve 122 such that a constant pressure may be maintained within the ACS120. For example, the ACS 120 may maintain a constant 37° C. temperatureand 5% carbon dioxide concentration. In some implementations, the ACS120 is an incubator.

Within the ACS 120 there are a plurality of ischemic model devices 110and cameras 130. The ischemic model device 110 is discussed further inrelation to FIG. 1B, but briefly, the ischemic model flow device 110 isa microfluidic flow device configured to model ischemic injuries. Thecameras 130 are placed such that they can view the cells within theischemic model device 100. In some implementations, the cameras 130 arepart of a microscope, and may be configured to capture fluorescenceimages of the cells. In some implementations, the camera 130 is placedoutside ACS 120 and views the ischemic model devices 110 and/or cellsthrough a window in the ACS 120. Access ports 121 are positioned aroundthe housing of the ACS 120 such that cables, connectors, and flow tubingcan pass into and out of the ACS 120 without disturbing the controlledatmospheric conditions.

As illustrated, system 100 includes at least one TER module 170. The TERmodule 170 is configured to measure the trans-endothelial electricalresistance across the cells in the ischemic model device 110. In someimplementations, trans-endothelial electrical resistance is a measure ofthe severity of damage caused by an ischemic event. In otherimplementations, the TER module 170 may be replaced or supplemented withother test equipment to monitor the cellular health within the ischemicmodel devices 110.

The system 100, as illustrated, also includes a plurality of fluid pumps140 and a fluid source 150. In some implementations, fluid from a fluidsource 150 may be forced through the ischemic model devices 110 by apump 140. The fluid source contains, in some implementations, atherapeutic agent as described below. In certain implementations, theoxygen levels with the fluid have been altered such that the fluidcauses a portion of the cells within the ischemic model device 110 tobecome ischemic.

FIG. 1B is an exploded view of the ischemic model device 110 discussedabove. The model 110 includes a chamber 117 in which cells (such asneurons) are cultured. Surrounding the chamber 117 is a chamber wall 118capable of containing cell culture medium. One side of the chamber (forinstance, the bottom) is formed by a porous barrier 116. Cells grown inthe chamber can adhere to the porous barrier 116. The porous barrier 116separates the chamber 117 from the base 112.

The base 112 includes three parallel channels 113, 114, and 115. Eachchannel has an inlet 113 a, 114 a, or 114 a through which fluid canenter, and an outlet 113 b, 114 b, or 115 b, through which fluid canexit. In some implementations, a user pumps fluid through the channelsusing a peristaltic pump. In some implementations, a different fluid ispumped through each channel. As illustrated, the base 112 does notcompletely enclose the channels; rather, the roof of each channel isprovided by the porous barrier 116.

In this arrangement, when a solution is pumped through the channels,solutes can pass through the porous barrier and reach the cells. Becausethe concentration of any solute may be highest near the channel throughwhich it is delivered, the cells closest to that channel may receive thehighest dose of that solute, while cells farther from that channel mayreceive lower doses. Channels can deliver solutes such as oxygenscavengers. Examples of oxygen scavengers are Na₂SO₃, sodium lactate,and EC-Oxidase. In some implementations, channels deliver oxygen, smallorganic molecules, macro molecules, and/or cells.

FIG. 2 illustrates a side view of a single channel ischemic model device200. The base 202 of the device has a single channel 204. Above the base202 and channel 204 is the porous barrier 210. Cultured cells adhere tothe barrier, and these cells can be divided into three categories basedon their position relative to the channel 204. The first population 216(also referred to as the “proximal cells”) is proximal to the channeland is shaded black. The second population 220 (also referred to as the“distal cells”) is distal to the channel and is shown in white. There isalso a population of cells 218 (also referred to as “intermediatecells”) shaded gray that lies between the proximal and distalpopulations. The cells are submerged in a solution 212 that allows thecells to remain viable at least over the course of an experiment.

In some implementations, the device 200 is used to create a twodimensional model of ischemia. For example, if the channel 204 is filledwith a high-oxygen fluid and the solution 212 has lower oxygen levels,the proximal cells 216 will receive high oxygen levels, the intermediatecells 218 will receive intermediate oxygen levels, and the distal cells220 will receive low oxygen levels. If, on the other hand, the channel204 is filled with a low-oxygen fluid or an oxygen scavenger, theproximal cells 216 will experience low oxygen levels, the intermediatecells 218 will experience intermediate oxygen levels, and the distalcells will experience high oxygen levels. In such a scenario, theproximal cells 216 would model the core of an ischemic stroke, theintermediate cells 218 would model the penumbra, and the distal cells220 would model the healthy tissue. In some implementations, the baselayer 202 includes multiple channels 204.

In some implementations, the proximal cells are within about 100 μm of achannel 204. In certain implementations, the proximal cells are withinabout 300, 200, 100, 75, or 50 μm of a channel 204. In certainimplementations, the distal cells are more than 200 μm, 500 μm, 1 mm, 2mm, or 5 mm from the channel 204. In some implementations, a cell isproximal to a channel 204 if the cell receives an effective dose of thesubstance being delivered by the channel.

The chamber walls of the device are made of a material suitable for cellculture. The material is strong enough to support the tissue culturemedia, non-toxic to cells, and substantially non-reactive with the othercomponents of the device. Examples of appropriate cell culture materialsinclude polymeric and/or non-polymeric materials, including PDAs,acrylic, polyethylene, polyolefin polymer, polyurethane, polystyrene,Pyrex, glass, polypropylene, or Penman.

The chamber is a suitable size for culturing cells. In someimplementations, the chamber accommodates a cell culture area of ˜0.5cm², close to the surface area of a section of adult rat brain. In someimplementations, the cell culture area is approximately 0.01-0.02 cm²,0.02-0.05 cm², 0.05-0.1 cm², 0.1-0.2 cm², 0.2-0.5 cm², 0.5-1 cm², 1-2cm², 2-5 cm², or 5-10 cm². The chamber walls are high enough to allowthe chamber to be filled with sufficient medium to support cellviability, growth, or metabolism.

The chamber can be filled with a liquid compatible with cell metabolismand/or growth. Suitable media for neuron al cultures include Dulbecco'smodified Eagle's medium supplemented with 10% (v/v) fetal calf serum,100 U/ml penicillin, and 0.1 mg/ml streptomycin and Neurobasal mediumsupplemented with B27 and 0.5 mM L-glutamine. One skilled in the artwill recognize that the chamber can be filled with other liquids. Insome implementations, during experiments it may be appropriate totemporarily culture the cells in a liquid that is free of macromolecules (an example is physiological saline solution (PSS)), even ifit does not support long-term cell growth. A liquid free of macromolecules can facilitate detection of factors that the cells secreteduring the experiment.

The amount of oxygen the solution 212 is exposed to is controlled by theACS 120 and/or other means. For example, the ACS 120 may maintain a lowoxygen concentration in the experimental chamber such that the onlyoxygen available to the cells is oxygen that diffuses through the poursbarrier 210 from the channel 204. Additional means of controlling theoxygen available to the cells may include placing a coverslip on thesurface of the liquid, closing a door in the device, or floating oil ontop of the liquid.

In certain implementations, the device does not require constantperfusion of the cell culture chamber. Avoiding perfusion can preventthe washout of factors, such as cytokines, that contribute to theextracellular milieu of the penumbra. In addition, avoiding perfusion ofthe neural cells avoids exposing these cells to shear stress. In certainimplementations, the cells are subject to shear stress forces of lessthan about 1, 0.5, 0.2, 0.1, 0.05, 0.02, or 0.01 dyne/cm².

The ischemic model should be maintained at a temperature compatible withcell viability, metabolism, and/or growth. Generally, incubation at 37°C. is appropriate for human cells, although in the case oftemperature-sensitive human cells, higher or lower temperatures can beused.

The base is be made of a material that is strong enough to support theweight of the cell culture and is capable of being shaped to producechannels. Appropriate materials for the base includepolydimethylsiloxane (PDAS), cyclic olefin copolymer (COC), andpolystyrene.

In some implementations, the porous barrier includes a membrane. Themembrane can serve as a substrate that supports the cells while allowingoxygen, oxygen scavengers, or other substances to travel from thechannels into the cell culture chamber.

In certain implementations, the barrier's pores are small enough toprevent the neural cells from substantially colonizing the channels. Insome implementations, however, the pores are large enough to allowimmune cells in the channels to enter the cell culture chamber. Inpreferred implementations, the average pore diameter is about 10 μm, forinstance between 8 μm and 12 μm.

The average pore diameter can also be about 0.4 μm, 1 μm, 2, μm, 3 μm, 5μm, 20 μm, or 50 μm, for instance within 20% of one of those diameters.In some implementations, the pore diameters are less than about 2 nm, 5nm, 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, or 400 nm. In someimplementations, the pore area constitutes about 7%, about 7-10%, orabout 5-15% of the surface area of the barrier.

The thickness of the barrier can vary with the application. In someimplementations, the barrier will have a thickness between 10 μm and 500μm. In some implementations, the barrier will have a thickness between100 nm and 500 μm. For instance, the barrier may be 100-200 nm, 200-500nm, 500 nm-1 μm, 1-2 μm, 2-5 μm, 5-10 μm, 10-20 μm, 20-50 μm, 50-100 μm,100-200 μm, or 200-500 μm.

The barrier should be compatible with cell metabolism and, optionally,cell growth. The barrier should also be one to which cells can adhere.Appropriate materials for the barrier include polycarbonate (PC),polyester (e.g., polyethylene terephthalate (PET)), collagen-coatedpolytetrafluoroethylene (PTFE), PDAS, polysulfone, and naturalelectrospun ECM proteins (such as collagen). In certain implementations,the barrier is non-biodegradable over time periods typical for cellculture in a given vessel. Certain polycarbonates, polyesters,polytetrafluoroethylenes, ethylene-vinyl acetates (EVA), and polyvinylacetates (PVA) are non-biodegradable.

Porous barriers can be produced by a number of methods. In someimplementations, a polymer is initially produced in a non-porous form,and pores are produced, for example by micromachining. Alternatively,the porous barrier can be produced using pore-forming agents or othersuitable techniques.

In some implementations the porous barrier is made from a differentmaterial from the base. However, in other implementations, the porousbarrier and the base are a single component. In such an instance, thechannels run through the middle of the base, and pores in the materialof the base are the conduits through which oxygen or other componentstravel to the cell culture chamber.

In some implementations, the cells adhere to the porous barrier. Incertain implementations, a significant number of, or substantially allof, the cells are in direct contact with the porous barrier.

The barrier may also contain functional components. In someimplementations, the barrier includes a moiety or component thatpromotes cell adhesion. This may be achieved with a proteinaceouscoating such as gelatin, fibronectin, or poly-lysine. The membrane mayalso contain an antibiotic and/or anti-fungal agent to inhibitcontamination, or one or more growth factors to promote cell growth anddivision.

The channels may be used to deliver oxygen to the cells. In someimplementations, a channel delivers a high amount of oxygen, forexample, an amount sufficient to maintain high cell viability over anextended period, modeling normoxic tissue. In some implementations, achannel delivers a low amount of oxygen, for example, an amountinsufficient for high cell viability over an extended period, modelinghypoxic or anoxic tissue such as the core or penumbra. The low amount ofoxygen may be less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%of the high amount of oxygen.

In some implementations, the device includes a single channel. In someimplementations, the device includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore channels. In some implementations, the channels run straight andparallel to each other.

However, in other implementations the channels are not straight. Theycan be designed with branching structures that mimic the capillariesthat supply the brain with oxygen. An example is shown in FIG. 3. Thebase 302 includes three channels 304, 306, and 308 that mimic the shapeof capillaries. The channels can have varying heights and depths.Capillary-like structures may be produced, for example, as described inBorenstein J et al. “Microfabrication Technology for Vascularized TissueEngineering”, Biomedical Microdevices 4(3) 167-175 (2002). Briefly,photolithographic techniques, injection molding, direct micromachining,deep RIE etching, hot embossing, or any combinations thereof can be usedto pattern the above described polymers. The pattern can then bemicromachined, for instance by silicon etching or a thick photoresistprocess. The micromachined wafers may be used as molds to producescaffolds with a feature resolution of about 1 micron. One appropriatematerial for the scaffolds is polydimethylsiloxane (PDAS). Additionalmethods for producing vasculature-like structures are described in USPatent Pub. No. 2007/0148139.

The barrier need not be flat. By recreating some of the structuralarchitecture of the brain, the cells populating the system willinherently be exposed to a more physiological environment. High aspectratio structures modeling the capillaries that oxygenate the brain canbe fabricated using standard fabrication and soft-lithographictechniques. Modified replication techniques can be utilized to transferthese structures into a porous barrier material.

In some implementations, the porous barrier includes a matrix such asMatrigel, collagen, hyaluronic-acid based gel, or agarose. A matrix maypromote formation of a three-dimensional cell culture.

Many of the devices described herein comprise fluid-filled channels thatare separated from the cell culture chamber. However, other designs arepossible. In some implementations, a device includes a single cellculture chamber which has inlets and outlets that allow a direct,laminar flow perfusion of differently oxygenated media through thechamber. For instance, the cell culture chamber may have two inlets andtwo outlets. One inlet may introduce oxygen-rich media into a first areaof the chamber, and the other inlet may introduce oxygen-poor media intoa second area of the chamber. Media flows through the chamber fastenough to keep the oxygenated medium from coming to equilibrium with thedeoxygenated medium.

II. Cell Components of the Ischemic Stroke Model

The devices herein can be used to model ischemic stroke conditions.Thus, in some implementations, the devices contain CNS cells. In simplerimplementations, the device contains a monoculture of a single celltype, for instance neurons. In more complex implementations, the devicecontains a mixture of CNS cell types. For instance, the culture maycontain neurons, astrocytes, and microglia. In some implementations, theculture contains neurons and at least one type of glial cell. The gliamay be microglia or macroglia. Macroglial cells include astrocytes,oligodendrocytes, ependymal cells, and radial glia. In someimplementations, the cells are neural precursors.

A user may control the ratio of different cell types in the device byselecting an appropriate cell culture medium and by controlling thetypes of cells introduced into the device. In some implementations, thecell ratios mimic that in the human brain or a rodent brain. In somepreferred implementations, the ratio of neurons to glia is about 3:7which is typical of the human brain. In some implementations, the ratioof neurons to glia is about 7:3 which is typical of the rat brain.

In some implementations, the cells are mammalian cells, for instancehuman, rat, or mouse cells.

In some implementations, the cultures achieve high levels of viabilityprior to the ischemic experiment, for instance over 80%, 90%, 95%, 98%,or 99% of the cultured cells may be viable.

In certain implementations, the device is capable of culturing cells forlong periods of time, such as at least 5, 10, 15, 20, 25, or 30 days. Insome implementations, the cultured cells exhibit functionalcharacteristics of neural cells, such as being GABAergic andglutamatergic.

In certain implementations, the device models one or morecharacteristics of the core and the penumbral region. In vivo, it isthought that neural cell death takes place in the core soon after theischemic event, but occurs at delayed time points at the penumbra. Thecore typically displays enhanced neuro-excitability and loss of membranepotential regulation, which lead to neuron al death. In the core regionof the ischemic insult, cells undergo anoxic depolarization andspreading depression, and the dying neurons may never repolarize. Inpenumbral regions, depolarizations are also observed. However, theseperi-infarct depolarizations are generally not sustained and therepolarization of neurons in the penumbra places further metabolicstress on cells in this region. There is some evidence from humanstudies that these peri-infarct depolarizations continue to occur 48-72hr following brain injury. In vivo, a direct correlation has beenobserved between the number of peri-infarct depolarizations and infarctsize. Another characteristic of ischemic neural tissue is the ubiquitoussecond messenger, Ca²⁺, which appears to be a point of convergence forvarious signaling pathways that result in cytotoxicity after stroke. Inaddition to promoting further membrane dysfunction, Ca²⁺ overloadcontributes to nitrosative and oxidative stress in cells by increasingthe activity of nitric oxide synthase and triggering mitochondrialdamage and concomitant production of nitrous oxide (NO) and reactiveoxygen species (ROS). The penumbra may also display elevated apoptosispathways. Thus, in some implementations, the ischemic model device isused to produce neural cells showing (for example) abnormal membranepotential regulation, calcium signaling, mitochondrial function, NOlevels, or ROS levels.

In allowing a user to subject different cells within a single culture todifferent oxygen levels, the devices herein allow a user to modelseveral important processes. For instance, a user can study theinteractions between cells subjected to different oxygen levels.Different populations of cells may interact when, for example, onepopulation secretes a factor that acts on the other population, or whendepolarizations in one population promote an energy imbalance in theother population. In certain implementations, the porous barrierseparating the channels from the cell culture chamber prevents thewash-out of soluble factors that mediate cross talk between normal,apoptotic, and necrotic cells observed in the penumbral region.Furthermore, in some implementations, the ischemic models herein modelthe transient peri-infarct depolarizations believed to occur in thepenumbra. In addition, in certain implementations, the models hereinallow a user to create significant pO₂ and glucose gradients observed inthe penumbra.

The CNS cells described above model the brain tissue that is affected byischemic stroke. Other cell types can be introduced into the device toprovide a more complex model.

One such cell type is endothelial. Under proper co-culturing conditions,neural and endothelial cells communicate with each other giving rise towhat is known as a “neurovascular unit,” a functional component ofneural tissue in vivo. Thus, to model a vascular unit, endothelial cellscan be introduced into the channels. The endothelial cells will adhereto the channel walls and regulate the passage of oxygen, smallmolecules, and macro molecules into the cell culture chamber. Across-section of such a device is illustrated in FIG. 4A.

In FIG. 4A, the device 400 includes a base 402 through which a channel404 runs. The channel is lined with endothelial cells 422. A porousbarrier 410 separates the base 402 from the cell culture chamber 412. Alayer of CNS cells 416 lies on top of the barrier 410. The endothelialcells 422 can block or actively transport substances from the channel404 into the cell culture chamber 412.

The endothelial cells 422 can be primary cells or established lines. Insome implementations, the cells are immortalized. In someimplementations, the endothelial cells form tight junctions. Tightjunctions are proteinaceous connections between adjacent cells, forminga barrier that is substantially impermeable to fluid. In tightjunctions, the membranes of adjacent cells are sealed together bymultiple redundant strands of transmembrane proteins, primarily claudinsand occludins. Tight junctions form naturally several contexts includingthe blood-brain barrier. Examples of appropriate endothelial cellsinclude HUVECs (human umbilical vein endothelial cells). Brain capillaryendothelial cell lines are also described in, for example, Hosoya et al.(“Conditionally Immortalized Brain Capillary Endothelial Cell LinesEstablished from a Transgenic Mouse Harboring Temperature-SensitiveSimian Virus 40 Large T-Antigen Gene” AAPS PharmSci. 2000; 2(3): article27) and Fasler-Kan et al. (“Cytokine signaling in the human braincapillary endothelial cell line hCMEC/D3” Brain Res. 2010 Oct. 1;1354:15-22). In certain implementations, the device is seeded withendothelial progenitor cells, which are then allowed or induced todifferentiate into mature endothelial cells. Exemplary endothelialprogenitor cells (EPCs) are described in von Ballmoos M et al.,“Endothelial Progenitor Cells Induce a Phenotype Shift in DifferentiatedEndothelial Cells towards PDGF/PDGFRβ Axis-Mediated Angiogenesis”, PLoSONE 5(11): e14107.

In certain implementations, muscle cells are also introduced into themodels described herein. In some sites (including proximal portions ofmetarterioles), blood vessels are flanked by smooth muscle cells thatregulate blood flow through the capillary. At the transition betweenmetarterioles and true capillaries, annular smooth muscle called aprecapillary sphincter typically regulates blood flow into thecapillary. Muscle cells can modulate capillary blood flow by contractingor relaxing and by secreting factors such as extracellular matrix,prostaglandins, and cytokines.

A third type of cell that can be introduced into the models herein is animmune cell 424. Immune cells 424 are a relevant part of an ischemicstroke model because they often migrate to the brain following ischemicinjury. During an ischemic event, inflammation within the brain and theblood vessels contributes to an ischemic injury. Secretion ofinflammatory cytokines directly attracts immune cells. In addition,although the blood-brain barrier usually protects the brain fromperipheral immune cells, ischemic stroke can disrupt the blood-brainbarrier and impair the integrity of the vascular units that provideoxygen to the brain. These events afford peripheral immune cells (suchas peripheral leukocytes and macrophages) entry into the brain. Certainimmune cells may cause damage by improperly attacking neural cells, andcertain immune cells may promote recovery by clearing necrotic cells.Thus, inclusion of immune cells in the device allows a user to model CNSdamage and recovery resulting from immune cells entering the neuraltissue.

In some implementations, the cells are chosen to mimic healthy tissue.In other implementations, the cells are selected as a model for adisease or a condition that exacerbates stroke. In some implementations,the cells comprise a mutation that increases the likelihood of a stroke(or a related condition) or the likelihood of serious injury once astroke occurs. Numerous disorders and mutations predisposing anindividual to a stroke are described in Szolnoki “Evaluation of theInteractions of Common Genetic Mutations in Stroke” Methods in MolecularMedicine, 2005, Volume 113, III, 241-249, DOI:10.1385/1-59259-836-6:241. In other implementations, the cells aremodified ex vivo to show such a disease phenotype. For instance, thecells may be treated with a toxin, subjected to RNA interference, orgenetically modified to overexpress or underexpress a gene of interest.

The device 400, in certain implementations, includes a first and secondlayer of electrodes, 432 and 430 respectively. The first layer ofelectrodes 432 is countersunk into the base layer 402 such that the topof the electrodes 432 do not raise above the top of the base layer 402.The top layer of electrodes 430 are deposited directly onto the underside of the roof of the chamber 412. In certain implementations, theroof 430 is a coverslip that is applied to the device 400 once the cellsa fully seeded into the device 400. The electrodes are between about 100μm and 150 μm in diameter. In certain implementations, the electrodesare stainless steel, platinum, chromium-gold alloys, or silver-silverchloride electrodes. In some implementations, each of the electrodes canact independently as a stimulation and/or recording site, such that atrans-endothelial endothelial resistance profile can be created alongthe path of the channel. In other implementations, the electrodes of aparticular layer are electrically coupled to one another, such that asingle trans-endothelial resistance is recorded for each channel. Insome implementations, the electrodes are used to record local fieldpotentials or other electrical activity. Trans-endothelial electricalresistance can be used as a measure of the integrity and/or health ofthe CNS cells 416. For example, the above described TER module 170 mayinduce an electrical pulse in the first layer of electrodes 432.Concurrently, the TER module 170 can record the resulting currentresponse at the second layer of electrodes 430. The TER module 170 thenintegrates the resulting current response to determine the impedance ofthe cell layer. In yet other implementations, the electrodes areconfigured as recording and/or stimulating electrodes.

FIG. 4B is an isometric view of the bottom layer 402 of device 400.Bottom layer 402 includes a plurality of electrodes 432 which partiallyoverhang channel 404. As illustrates, the electrical leads of electrodes432 converge onto a single trace 433. As discussed above, in someimplementations, the electrodes do not converge onto a single trace 433such that the electrodes can be independently accessed.

III. Methods of Using the Ischemic Stroke Model

FIG. 5 is a flow chart illustrating a method 500 for modeling ischemicstroke. Further method examples are provided in Sections III (A), (B),(C), and (D), but briefly, the method of modeling ischemic stroke beginsby providing a microfluidic flow device (step 501). A population ofcells is then seeded into the microfluidic flow device (step 502). Afirst portion of the population of cells is exposed to a lowconcentration of oxygen (step 503) and a second portion of thepopulation of cells is exposed a high concentration of oxygen (step504). The population of cells are then exposed to a test agent (step505). Finally, at least one cellular property is measured (step 506).

As set forth above, and referring to FIG. 1B, a microfluidic flow deviceis provided (step 501). As discussed above, the microfluidic flow devicecan be a single or multi-channel flow device. The at least one channelof the microfluidic flow device is separated from a cellular chamber bya porous membrane. In some implementations, the porous membrane isconfigured such that the cells in the cellular chamber do not experiencea shear force as fluid flows through the at least one channel. In someimplementations, the cellular chamber is sealed to reduce the cellsexposure to oxygen or other gasses.

Next, a population of cells is seeded into the microfluidic flow device(step 502). In some implementations, the cells are from the centralnervous system, and can include such cells as neurons, microglia,astrocytes, oligodendrocytes, or neural progenitors. In certainimplementations, the cells are dissociated neural cells and in otherimplementations the cells comprise an acute brain slice.

The first portion of the cells is exposed to low concentrations ofoxygen (step 503) while a second portion of cells is exposed to higherconcentrations of oxygen (step 504). As described above, the there are anumber of ways to create an oxygen gradient across the cells in themicrofluidic flow device. For example, an increase in oxygenconcentration proximal to the channel can be created by flowing oxygenrich fluid through the flow channel. Alternatively, a decrease in oxygenconcentration proximal to the channel can be created by flowing oxygenscavengers through the flow channel. The oxygen gradient is designedsuch that the lack of oxygen creates an ischemic state within the firstportion of cells. In some implementations, the fluid in the cellularwell can be augmented with oxygen or oxygen scavengers. In certainimplementations, there is a third portion of the cellular populationlocated with the region of the cellular well that transitions from lowto high oxygen concentrations. In some implementations, the microfluidicflow device is placed into an atmospheric control system, such as theACS 120, which further controls the amount of oxygen to which the cellsare exposed.

As described below in relation to FIG. 6, the cells are exposed to atest agent (step 505). In some implementations, the test agent is achemical therapeutic that promotes neural cell survival, inhibitsneurodegeneration, inhibits necrosis, or inhibits apoptosis. In otherimplementations, the test agent may be high concentrations of oxygen,cells (e.g., stem cells), or electrical stimulation.

Next, at least one cellular factor is tested (step 506). Differentfactors can be tested in relation to different biochemical studies,microscopy studies, and drug screening studies. For example, in someimplementations, the testing step may include testing for a specificchemical secreted by the cells during experimentation, measuringtrans-endothelial electrical resistance, measuring cellularfluorescence, or any combination thereof. Below, several examples areprovided to further illustrate modeling ischemic stroke with the abovedescribed microfluidic device.

A. Biochemical Studies

The ischemic stroke model devices herein may be used to study thebiochemistry of ischemic regions. To that end, the model allows a userto collect samples of cells or fluid that has contacted the cells. Insome implementations, a user takes a sample of cells from a regionmodeling the core and/or penumbra, and compares them to cells from aregion modeling normoxic tissue.

In other implementations, a user can withdraw an aliquot of fluid fromthe cell culture chamber. This aliquot can be tested for the presence offactors secreted by the cells.

In still other implementations, the user withdraws a sample fromeffluent from a channel. Because the barrier is porous, it is expectedthat factors secreted by the cells will enter the channels. Samplingeffluent from the channels has the advantage of not disturbing thetissue culture chamber. Thus, the device permits dynamic sampling ofperfusates or effluents from discrete areas in the culture. Thesesamples can be tested for the presence of (for example)neurotransmitters and cytokines.

The device can also be used to observe the cells' response to anexogenous substance. The exogenous substance can be added to the cellculture chamber or one or more of the channels. For instance, in someimplementations, cytokines are added to stimulate the inflammation thatcan accompany ischemic stroke in vivo. Appropriate cytokines for thispurpose include TNF-a and IL-1β.

B. Microscopy

The ischemic model can be designed to permit microscopy of the culturedcells. Monitoring the cells by microscopy allows long-term, non-invasiveanalysis of stroke progression over a physiologically relevant timecourse. If the device is sufficiently transparent, it can be placed on amicroscope stage and inspected using light microscopy. Generally, thebase, barrier, and cell culture medium should be sufficientlytransparent to allow light to pass through the portions of the devicewhere the cells are cultured. PDAS is one optically transparentmaterial. In some implementations, the device allows at least 90%, 95%,98%, or 99% of light of a given wavelength to pass through components ofthe device disposed along a path that is perpendicular to the porousbarrier and that passes through the cells.

Fluorescence microscopy may also be used. Numerous relevant fluorescentdyes are available for monitoring, e.g., membrane potential, pH, andfree Ca²⁺ levels. In addition, the localization and/or levels ofspecific enzymes can be monitored using fluorescent fusion proteins. Insome implementations, multiple analytes are detected at once usingfluorophores with compatible emission and excitation spectra.

To be suitable for fluorescence microscopy, the device should besufficiently transparent and have little or no fluorescence whenilluminate with at least one wavelength. In some implementations, thedevice is substantially non-fluorescent in response to far-red, red,orange, yellow, green, blue, violet, or ultraviolet light. If the cellculture chamber is sealed, it may be sealed using glass which is UVcompatible and permits quantitative fluorimetry.

The slimness of the entire device can be attuned to be compatible withhigh resolution imaging for high content analysis. In someimplementations, the microchannel thickness can be varied (using spincoated and alaminated layer of PDAS) from 100 μm-2 mm, a large spectrumthat can facilitate high resolution microscopy for high contentanalysis.

C. Drug Screening

One major use for the devices herein is screening therapeutics. Thetherapeutic may be, for instance, a small molecule, a macromolecule, ora cell-based therapeutic such as a stem cell or an immune cell. Thetherapeutic may be, for instance, an agent that promotes neural cellsurvival, inhibits neurodegeneration, inhibits necrosis, or inhibitsapoptosis.

The test therapeutic may be added directly to the cell culture chamberor to one or more of the channels. In some implementations, thetherapeutic is a cell and it is added to the channel so that it musttraverse the porous barrier before reaching the neural cells. In someimplementations, the channels comprise a layer of endothelial cells, andthe therapeutic must traverse the endothelial cells before reaching theneural cells.

Drug screening is often performed in a high-throughput manner, and thedevices herein are amenable to high-throughput screening. FIG. 6illustrates three devices 626, 628, and 630 arranged in parallel. Eachdevice has three channels so that each device can apply three differentsolutions to different regions of cells. Three sets of tubing 632, 634,and 636 link the channels to three peristaltic pumps 638, 640, and 542.Each of the devices in FIG. 6 could contain a different test agent. Insome implementations, a device contains no test agent or it contains acontrol agent with a known effect. In some implementations, theperistaltic pumps deliver test agents to the cells via the channels. Forsimplicity, FIG. 6 only depicts three devices; however, the number couldreadily be scaled up.

In contrast to FIG. 6 which arranges the devices in parallel, thedevices could be arranged in series such that fluid flows through achannel in one device and then into a channel in the next device and soon. Such a setup can be arranged so that every device in the seriesreceives substantially the same concentration of oxygen or other factorfrom the channels. To accomplish this uniformity, the channels should besufficiently large and the concentration of oxygen or other factor inthe channel should be sufficiently high that it is not exhausted by thetime it reaches the final device in the series.

D. Uses for Modeling Conditions Other than Ischemic Stroke

The devices herein, though often referred to as ischemic stroke models,can also be used to model other conditions in which two or more nearbypopulations of cells, e.g., populations along a diffusion gradient, areexposed to different environments. In certain implementations, thedevices are used to model the communication or interaction between thetwo or more populations of cells through, e.g., diffusible signals.

First, the devices can be used to model neuron al injury. Typicalsources of neuron al injury include chemical toxicity, neurodegenerativediseases such as multiple sclerosis and Amyotrophic lateral sclerosis,angiogenesis (for instance when triggered by stroke), and certain formsof metastasis in which cancer cells invade the CNS. In someimplementations, the agent that induces the injury is introduced intothe chamber, and in some implementations, the agent is introduced intoone or more channels.

Second, the devices can be used to model ischemia or hypoxia innon-neuron al cell types. For instance, solid tumors are often hypoxicand this model can replicate the hypoxic condition. The devices can alsobe used to model ischemia in cardiac muscle tissue.

Third, the devices can be used to model basic neuron al cell functions.For instance, they can be used to model axonal guidance for central andperipheral nervous system regeneration research. The devices can also beused to study cell migration during development and stem cell biologyand cell invasion during wound healing and inflammatory conditions.

Fourth, the devices can be used to model numerous infectious diseasesthat affect the brain. Examples include HIV, bacterial meningitis,cerebral malaria, prion diseases, Ebola, hantavirus, and hemorrhagicfevers.

Fifth, the devices can be used to model cell therapies. Following aninjury (for example, a neuron al injury as described above), a usercould introduce a test cell into the device to determine the test cell'seffect on the injured cells. For instance, the user could model the testcells' effect on the blood-brain-barrier and/or neural repair(neurogenesis). In some implementations, the test cell is a stem cell, acord blood cell, or a progenitor cell. In some implementations, the testcell is administered to the fluid-containing chamber, and in someimplementations, the test cell is administered into one or morechannels.

Sixth, a user can study “bystander effects” using the devices herein.The bystander effect is a phenomenon in which a therapeuticallyadministered cell affects nearby endogenous cells, for instance bysecreting factors such as growth factors or by taking in factors fromthe cellular milieux. In some implementations, the therapeutic cellpromotes neuron al repair. In some implementations, the therapeutic cell(such as a stem cell, a progenitor cell, or myeloid cell) drives outamyloid beta from the neural side to the vascular side.

IV. Kits

In some implementations, the device is provided as a kit that a user canassemble. The kit may include (a) a chamber wall capable of delineatinga cell culture chamber that contains tissue culture cells and medium,(b) a base comprising a channel, wherein the channel has an inlet and anoutlet, and the channel is sized to be proximal to a first region of thechamber and distal from a second region of the chamber; (c) amicroporous membrane suitable as a substrate for cell culture, whereinthe microporous membrane is sized to separate the channel from thechamber. The kit may also include means for securing the microporousmembrane between the chamber and the channel. The means for securing themicroporous membrane between the chamber and the channel may be, forinstance, one or more of: adhesive, screws, clamps, a waterproofsealant, and an interlocking assembly such as a raised portion that fitssecurely into a cavity.

EXAMPLES I. Fabrication of a Microfluidic Cell Culture Device

A microfluidic device for subjecting different neural cells to differentenvironments can be produced according to the methods below. This devicebears a neural compartment juxtaposed above three parallelmicrochannels. The neural compartment is separated from themicrochannels by a microporous polyester membrane bonded irreversiblythrough a thin layer of silicone adhesive. FIG. 1 shows an exploded viewof such a device.

The neural chamber accommodates a cell culture area of ˜0.5 cm², closeto the surface area of a section of adult rat brain. The channels arefabricated to have dimensions of 1 mm width, 250 μm height and 1.1 cm(length) separated from each other by 1 mm. The design of the neuralchamber facilitates standard well-plate format of cell culture, which issimple, routine, and does not require continuous perfusion. Thereservoir in the culture chamber holds ˜150 μl media. The membrane (10μm thickness and pore size 8 μm) in between the neural chamber and themicrochannels acts as an efficient mechanical barrier to prevent directexposure of shear to neural cells and washing out of necessary factors(a significant advantage over focal injury model). The sides of thedevice can be sealed with silicone glue or epoxy to minimize O₂diffusion from bulk (as PDAS is permeable to oxygen). Prior to a hypoxicexperiment, the cell culture media in the neural chamber may be replacedby physiological saline solution (PSS) and sealed using a glasscoverslip to limit O₂ diffusion from ambient environment. PSS generallycontains 140 mM NaCl, 1.2 mM MgCl₂, 3 mM KCl, 2.5 mM CaCl₂, 7.7 mMglucose, and 10 mM HEPES, and is set to a pH of 7.4 with NaOH.

The flow of separately oxygenated media at optimized flow rates (in asealed device) were modeled computationally using COMSOL Multiphysics™software chemical engineering module (FIG. 7). The computation modelsthe steady state levels of oxygen in the device, without oxygenconsumption.

II. Use of a Microfluidic Device for Studying the Effects of an IonGradient on Neural Cells

A microfluidic device was fabricated. This device had a neural chamberjuxtaposed above two parallel microchannels of dimensions 1 cm(length)×800 μm (width)× and 100 μm height. The channel layer wasfabricated using soft lithographic molds and cast with PDAS pre-polymer.The neural chamber was a molded block of PDAS polymer punched out toaccommodate a cell culture area of ˜0.35 cm². A microporouspolycarbonate (PC) membrane with 10 μm pores separates the chamber fromthe microchannels. The two casted layers were bonded to the PC membraneusing a thin uniform layer of silicone glue (3140, Dow). FIG. 8 is aphotograph of the device where one channel is filled with green dye andthe other with orange dye.

Following device assembly, the chips were sterilized with ethylene oxideand the neural chamber was coated with poly-D-lysine (50 μg/ml)overnight at 37° C. Rat cortical neural cells isolated from embryonicday 18 Sprague-Dawley pups were then seeded into the neural chamber at adensity of 105 cells/cm². Methods for culturing neurons derived from ratembryos are described in Katnik et al. (“Sigma-1 Receptor ActivationPrevents Intracellular Calcium Dysregulation in Cortical Neurons duringin Vitro Ischemia” J Pharmacol Exp Ther. 2006 December; 319(3):1355-65).The cells were differentiated into a predominant mixture of neurons,astrocytes, and microglia following 10 days of culture in Neurobasal™medium supplemented with 2 mM L-glutamine, 10% v/v fetal bovine serum,serum free B27, 10 ng/mL basic fibroblast growth factor (FGF), and 1%v/v penicillin/streptomycin.

Immunohistochemical staining provided evidence that the cells haddifferentiated, and that neuron had formed synaptic contacts and wereactively synthesizing neurotransmitters (FIG. 9). The top three panelsof FIG. 9 show, from left to right, neuron al cells identified using themarker MAP2, astrocytes identified using the marker GFAP, and microgliaidentified using the marker Ox-42. The bottom three panels of FIG. 9 aremerged images where DNA is shown in blue and immunofluorescence signalis shown in the red and green channels. From left to right, GABA isshown in green and MAP2 is shown in red, glutamate is shown in green andMAP2 is shown in red, and tubulin is shown in green, and synapsin isshown in red. The viability of these cultures exceeded 95% after 10days. A typical cell-type ratio is 20-30% neurons, 65-80% astrocytes,and 2-5% microglia. Of the microglia, typically more than 75% areresting or ramified.

The neural cells in the microfluidic chamber were studied using Ca2+fluorometry with fura2-AM. Basal levels of [Ca2+]i are represented asred in the image pseudo color, regions of elevated [Ca2+]i are blue. Thetop of the field of view was oriented along the inside edge of one ofthe microchannels. Just prior to acquiring the first image the normalphysiological saline solution (PSS) flowing through the channel waschanged to PSS containing 30 mM K+. Delivery of high K+ via themicrochannel triggered a [Ca2+]i wave (area outlined by dashed line inimages in FIG. 10A). The change in [Ca2+]i reveals, first, that there issufficient exchange between the microchannel and the culture compartmentto produce a change in K+ and elicit a cellular response. Second, thereis decremental conduction of the signal and the effect of high K+ istemporally and spatially limited (FIGS. 10A-10B). Third, the modelallows a user to effectively record from a broad area of the chip suchthat responsive and non-responsive areas can be simultaneouslymonitored. Thus, data obtained with this model suggests that the modelcan successfully generate a discrete area of ischemia.

III. Establishment of O₂ Gradients Using an Ischemic Model Device

The first step in obtaining a simulated core and a penumbral region inthe microdevice will be to obtain suitable oxygen gradients in thepresence of differentiated neural cells. The level of oxygen in thedevice can be measured using a glass insulated platinum needle (˜30 μmin diameter) connected to a Polarographic Amplifier (A-M Systems Model1900). A polarization voltage of 20.6 V can be used. At this voltage,the current output is proportional to the concentration of dissolved O2.To enable access to the chamber by the electrode, the coverslip which isotherwise used to seal the chamber can be replaced with a layer ofoxygen impermeable paraffin oil.

Oxygen gradients can also be varied by adjusting the followingvariables. First, the number or dimensions of the channel can bealtered. Second, the height (and therefore the volume) of the neuralculture can be altered. Third, the flow rate of the solution through themicrochannels can be varied. In some implementations, multipleperistaltic pumps are used to provide a different flow rate throughdifferent microchannels. Fourth, one can select one or more methods forincreasing or decreasing O₂ concentration. For instance, one can bubblePSS with N₂ to eliminate O₂ in the ischemia chamber. As another example,one can add an O₂ scavenger to the chamber or a channel. One example ofan O₂ scavenger is Na₂SO₃, which may be used at 500 μM on culturedcells. In some implementations, the device has three channels, and thechannel corresponding to the normoxic region contains fluid with 95%oxygen, the channel corresponding to the penumbra region has 5% oxygen,and the channel corresponding to the core has 0% oxygen.

Because an important function of oxygen is promoting glucose metabolism,the effects of hypoxia can be amplified by lowering the levels ofglucose to which the cells are exposed. For instance, glucose may bereduced to 2 mM in the PSS for the penumbra channel and to 0 mM in thecore channel.

IV. Detecting Apoptosis and Necrosis in Cells Subjected to High or LowOxygen Levels in an Ischemic Model Device

The devices herein allow a user to quantify the number of healthy,apoptotic, and necrotic cells at select locations relative the channelsin the device. From the oxygen gradient production condition obtained inExample III, one may utilize a Promokine™ Kit to quantify thedistribution of apoptotic, necrotic, and healthy cells in the device.Apoptotic cells can be identified with fluorescein-labeled Annexin V(green). Necrotic cells can be labeled with ethidium homodimer (red).All cells can be counterstained with Hoechst (blue). Following staining,the cells can be fixed with 2% formaldehyde and visualized using filtersets for FITC, rhodamine and DAPI. Cells appearing green (with blue),red (with blue), and blue only can be labeled as apoptotic, necrotic,and healthy, respectively. Five different sections along the length ofeach microchannel will be manually counted and quantified. Subsequent toquantification, the death rates can be compared with historical animaldata from in vivo experiments. The parameters outlined in Experiment IIIcan be used to produce a neural cell culture with necrosis and apoptosislevels typical of core, penumbra, and healthy tissue.

Apoptosis and necrosis can be detected according to the followingprotocols. One may use the Invitrogen Vybrant Apoptosis Assay Kit #6 orcombination of annexin V Alexa Fluor® 350 conjugate and propidiumiodide. Both the assay kit and the alternative stain have a fluorescentspectrum compatible with di-8-ANEPPS, fura-2 and DAF-FM and so can beused in combination with these indicators. In this and otherexperiments, fluorometry data can be compared with apoptosis/necrosismarkers using the microchannels and the edge of the chamber aslandmarks.

V. Detecting Electrical Activity in Neurons Subjected to High or LowOxygen Levels in an Ischemic Model Device

The devices herein allow a user to study neuron depolarization underischemic conditions. The voltage-sensitive dye, di-8-ANEPPS, can be usedto monitor electrical activity in cell cultures within the microfluidicchamber before and during oxygen-glucose deprivation (OGD) using theparameters set out in Example III to produce the desired normal,penumbra and core regions. Cells can be imaged using a Zeiss Fluar10×0.50 objective (23 mm field of view). One may sample at acquisitionrates ranging from 0.033 Hz to 1 Hz to determine what is the minimumrate that permits detection of the onset and extent (spatial andtemporal) of spreading depression (SD) and peri-infarct depolarizations(PIDs). The higher sampling rate (1 Hz) is sufficient to permits use offluorescent imaging techniques to record bursts of electrical activityin cortical neuron cultures. However, for extended recordings (>1 hr),lower sampling rates may be preferable to reduce photobleaching of thevoltage-sensitive dye. These lower rates will still permit monitoring oflong-lived changes in membrane potential, such as those associated withSD and PID. The sampling can be varied within a single experiment todetermine how the SD and PID affect bursting behavior. In someexperiments one can evoke depolarization within the normal zone byadding high K+ via the microchannel to determine if depolarization ofcells in this region affects membrane responses in the penumbra or core.Similarly, the response to reperfusion can be determined.

The voltage sensitive dye di-8-ANEPPS can be used according to thefollowing protocol. Cells can be incubated in 2 μM di-8-ANEPPS (10 mMstock solution in DMSO) for 20 minutes at room temperature. Cells can beilluminated at 450 nm and emission intensity >570 nm can be recordedusing SlideBook software. The typical sensitivity of di-8-ANEPPS tomembrane potential changes is 1% .F/F for 100 mV.

In this and other fluorometry experiments, the fluorometry data can beanalyzed as follows. The data can be converted to text files andimported into Clampfit 9 for analysis. For ROS fluorometry, fluorescenceintensity can be analyzed using non-linear or linear regression, asappropriate. The rate of change in fluorescence (.F/min) can becalculated before, during and following each condition. Statisticalanalysis can be conducted using SigmaPlot 9 and SigmaStat 3. Statisticaldifferences can be determined using paired and unpaired t-tests forwithin group and between group experiments, respectively, and will beconsidered significant if P<0.05. multiple group comparisons one may useeither a 1-way or a 2-way ANOVA, as appropriate. When an ANOVA indicatessignificant difference, one may use a Tukey Test to determinesignificance between groups. For all experiments one may conduct a Poweranalysis with SigmaStat 3.

VI. Studying Ca²⁺ and NO Signaling in Neurons Subjected to High or LowOxygen Levels in an Ischemic Model Device

The devices herein allow a user to study neuron al Ca²⁺ signaling underischemic conditions. An oxygen gradient can be produced according toExample III. Ca²⁺ levels can be assayed using fura2 as described inExample II. The magnitude and spatial and temporal patterns of [Ca²⁺]ichanges evoked by OGD can be determined. Changes in [Ca²⁺]i can becompared with the distribution of apoptotic and necrotic cells. By usingboth di-8-ANEPPS with fura-2, a user can determine the correlationbetween membrane potential changes and [Ca²⁺]i dyshomeostasis.

Using parameters for OGD and reperfusion duration according to ExampleIII, neuron al cultures can be subjected to localized ischemia andreperfusion. To detect NO levels, neurons can be loaded with theNO-indicator dye DAF-FM and imaged at 0.033 Hz. Alternatively or incombination, cells can be loaded with DHE imaged at 0.033 Hz to measureROS production. In addition, cells can be loaded with both fura-2 andDAF-FM. Simultaneous Ca²⁺ and NO fluorometry can be carried out todetermine the correlation between changes in the cellular levels ofthese molecules in response to ischemia. Apoptosis and necrosis can beassayed simultaneously using the Invitrogen Single Channel AnnexinV/Dead Cell Apoptosis Kit, which uses fluorescence intensity in thegreen channel to identify apoptotic and necrotic cells.

To measure the rate of NO and O₂-production, cortical neurons can beloaded by incubating for 1 hr at 23° C. in 5 μM DAF-FM or for 10 min at23° C. in 2 μM DHE (both in PSS, 0.1% DMSO). For DAF-FM and DHE theexcitation wavelengths can be 480 nm and 515 nM, respectively, andemission light can be collected at 520 nm and 605 nm, respectively. Thesame hardware and software used for Ca²⁺ imaging can be used for theseexperiments. One may monitor both [Ca²⁺]i and NO by alternatingillumination of the sample with excitation of 350, 380, and 490 nmsequentially and collecting emitted light at 510 nM. The sampling ratesmay be 0.33 Hz, 0.033 Hz for 30 min and 6 hr experiments, respectively.

VII. Studying Neuron-Secreted Factors in an Ischemic Model Device

The devices described herein allow a user to sample effluents fromdiscrete regions of the chamber as well as from the channels. Forinstance, a user may collect the effluent from each microchannel before,during and after exposure to OGD. The user may then measure the levelsof cell-secreted factors (such as IL1 IL10 and TNF-a) in the perfusatescollected from normal, penumbral and core regions following disruptionof pO₂ and glucose delivery. Using the device, one may measure cytokinesand specific neuromodulators or neurotransmitters such as glutamate andATP in the effluent.

The secreted factors may be tested using a variety of assays such asELISA, Western blot, capillary zone electrophoresis (CZE) orelectrochemiluminiscence. If the ELISA assay is used, collected effluentcan be centrifuged for 10 minutes at 1200 g followed by 0.2 μmfiltration to remove particulate matter. Solutions can be analyzed forthe presence of cytokines using ELISA Assays Kits (Invitrogen). Atypical sample sizes is 50 μl, and typical detection levels are: IL-1B,31-2000 pg/ml; IL-10, 15.6-1000 pg/ml; TNFa, 15-1000 pg/ml. Theseparticular cytokines are of interest because they have been associatedeither with pro-inflammatory (IL-1 and TNF-a) or 15 anti-inflammatory(IL-10) activity during stroke.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually 20 indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific implementations of the subject invention have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification and the claims below. Thefull scope of the invention should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations.

What is claimed:
 1. A method of modeling an ischemic condition,comprising: providing a microfluidic device including a fluid-filledchamber, a channel, a porous barrier separating the chamber from thechannel, a first population of CNS cells proximal to the channel, and asecond population of CNS cells distal from the channel relative to thefirst population of CNS cells; concurrently with exposing the firstpopulation of CNS cells to a low concentration of oxygen, exposing thesecond population of CNS cells to a high concentration of oxygen,thereby modeling the ischemic condition, wherein: exposing the firstpopulation of CNS cells to a low concentration of oxygen comprisesflowing an oxygen scavenger through the channel; and measuring at leastone cellular property of the first population of CNS cells.
 2. Themethod of claim 1, wherein ischemic condition is ischemic stroke.
 3. Themethod of claim 1, wherein the CNS cells comprise neurons, microglia,astrocytes, oligodendrocytes, or neural progenitors.
 4. The method ofclaim 1, further comprising producing a third population of CNS cellsbetween the first population and the second population, wherein thethird population of CNS cells models a penumbra produced by an ischemicstroke.
 5. The method of claim 1, further comprising measuringtrans-endothelial electrical resistance across the first and secondpopulation of cells.
 6. The method of claim 1, further comprisingvisualizing the cells in the device by microscopy.
 7. The method ofclaim 1, further comprising removing the cells from the device andperforming biochemical analysis or microscopy on the removed cells. 8.The method of claim 1, wherein measuring the at least one cellularproperty further comprises testing for a factor secreted by the cells.9. The method of claim 8, comprising measuring for the factor in asample taken from the chamber.
 10. The method of claim 8, comprisingmeasuring for the factor in a sample taken from the channel.
 11. Themethod of claim 1, further comprising exposing the cells to a testagent.
 12. The method of claim 11, wherein the test agent promotesneurodegeneration, necrosis, or apoptosis.
 13. The method of claim 11,wherein the agent is a cancer cell capable of invading neural tissue.14. The method of claim 1, further comprising not perfusing a fluidthrough the fluid-filled chamber.
 15. A method of modeling an ischemiccondition, comprising: providing a microfluidic device including afluid-filled chamber, a channel, a porous barrier separating the chamberfrom the channel, a first population of CNS cells proximal to thechannel, and a second population of CNS cells distal from the channelrelative to the first population of CNS cells; concurrently withexposing the first population of CNS cells to a high concentration ofoxygen, exposing the second population of CNS cells to a lowconcentration of oxygen, thereby modeling the ischemic condition,wherein: exposing the first population of CNS cells to a highconcentration of oxygen comprises flowing oxygenated fluid through thechannel; and measuring at least one cellular property of the firstpopulation of CNS cells.